Article

Context-Dependent and Disease-Specific Diversity

in Protein Interactions within Stress Granules
Graphical Abstract Authors
Sebastian Markmiller, Sahar Soltanieh,
Kari L. Server, ..., Eric J. Bennett,
Eric Lécuyer, Gene W. Yeo

Correspondence
geneyeo@ucsd.edu

In Brief
Interactions between stress granule
proteins exist ahead of a stress response
and candidate SG proteins modify
disease phenotypes in vivo.

Highlights
d APEX proximity labeling reveals 150 unknown SG proteins
in a dense protein network

d SG composition varies by stress and cell type, especially in

neuronal cells

d ALS motor neurons contain SGs with distinct content and

subcellular distribution

d SG proteins modify ALS-mutant-mediated toxicity in fly

models of neurodegeneration

Markmiller et al., 2018, Cell 172, 590–604

January 25, 2018 ª 2017 Elsevier Inc.
https://doi.org/10.1016/j.cell.2017.12.032
 Article

Context-Dependent and Disease-Specific Diversity

in Protein Interactions within Stress Granules
Sebastian Markmiller,1,2,3 Sahar Soltanieh,4 Kari L. Server,1,2,3 Raymond Mak,5 Wenhao Jin,6 Mark Y. Fang,1,2,3
En-Ching Luo,1,2,3 Florian Krach,1,2,3 Dejun Yang,7 Anindya Sen,8 Amit Fulzele,5 Jacob M. Wozniak,9,10
David J. Gonzalez,9,10 Mark W. Kankel,8 Fen-Biao Gao,7 Eric J. Bennett,5 Eric Lécuyer,4,11,12 and Gene W. Yeo1,2,3,6,13,14,*
1Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093, USA
2Stem Cell Program, University of California, San Diego, La Jolla, CA 92093, USA
3Institute for Genomic Medicine, University of California, San Diego, La Jolla, CA 92039, USA
4Institut de Recherches Cliniques de Montréal, Montréal, QC H2W 1R7, Canada
5Division of Biological Sciences, University of California, San Diego, La Jolla, CA 92093, USA
6Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117593, Singapore
7Department of Neurology, University of Massachusetts Medical School, Worcester, MA 01605, USA
8Neuromuscular & Movement Disorders, Biogen, Cambridge, MA 02142, USA
9Department of Pharmacology, University of California, San Diego, La Jolla, CA 92093, USA
10Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA 92093, USA
11Département de Biochimie et Médecine Moléculaire, Université de Montréal, Montréal, QC H3C 3J7, Canada
12Division of Experimental Medicine, McGill University, Montréal, QC H3A 1A3, Canada
13Molecular Engineering Laboratory, A*STAR, Singapore 138673, Singapore
14Lead Contact

*Correspondence: geneyeo@ucsd.edu
https://doi.org/10.1016/j.cell.2017.12.032

SUMMARY form ribonucleoprotein particles (RNPs). Large numbers of

Stress granules (SGs) are transient ribonucleoprotein
(RNP) aggregates that form during cellular stress and
are increasingly implicated in human neurodege-
neration. To study the proteome and compositional
diversity of SGs in different cell types and in the
context of neurodegeneration-linked mutations, we
used ascorbate peroxidase (APEX) proximity labeling,
mass spectrometry, and immunofluorescence to
identify 150 previously unknown human SG compo-
nents. A highly integrated, pre-existing SG protein
interaction network in unstressed cells facilitates
rapid coalescence into larger SGs. Approximately
20% of SG diversity is stress or cell-type dependent,
with neuronal SGs displaying a particularly complex
repertoire of proteins enriched in chaperones and
autophagy factors. Strengthening the link between
SGs and neurodegeneration, we demonstrate aber-
rant dynamics, composition, and subcellular distribu-
tion of SGs in cells from amyotrophic lateral sclerosis
(ALS) patients. Using three Drosophila ALS/FTD
models, we identify SG-associated modifiers of
neurotoxicity in vivo. Altogether, our results highlight
SG proteins as central to understanding and ulti-
mately targeting neurodegeneration.
INTRODUCTION
Cellular RNA molecules interact with a diverse array of nearly
2,000 RNA-binding proteins (RBPs) (Brannan et al., 2016) to
RNPs frequently accumulate into microscopically visible RNP
granules, which can measure up to several microns in size but
remain highly dynamic. Neuronal transport granules, which
mediate the transport of mRNA and other cargo along axons
and dendrites (Ainger et al., 1993), are an example of RNP gran-
ules that are present in cells under physiological conditions. By
contrast, exposure of cells to exogenous stresses can induce
the rapid formation of cytoplasmic stress granules (SGs) and
other induced RNP granules (Kedersha et al., 1999). The forma-
tion of SGs occurs concurrently with alterations in global RNA
metabolism, primarily a near-complete shutdown in translation
through sequestration of untranslated mRNAs within stalled
translation initiation complexes (Kedersha and Anderson, 2002).
SG formation has been suggested as a two-step process, with
initial formation of a dense stable SG ‘‘core’’ followed by accu-
mulation of proteins containing intrinsically disordered regions
(IDRs) and low-complexity domains (LCDs) into a peripheral
‘‘shell’’ through a process involving liquid-liquid phase separa-
tion (LLPS) (Jain et al., 2016; Molliex et al., 2015; Wheeler
et al., 2016). Recently, SGs have been associated with human
neurodegenerative disorders characterized by the presence of
toxic insoluble protein aggregates. This link is most compelling
in the case of amyotrophic lateral sclerosis (ALS) and frontotem-
poral dementia (FTD), where numerous disease-causing muta-
tions are purported to interfere with LLPS-dependent growth
and dynamics of SGs (Boeynaems et al., 2017; Lee et al.,
2016; Lin et al., 2016; Mackenzie et al., 2017; Martinez et al.,
2016; Murakami et al., 2015; Patel et al., 2015). A more complete
description of how SG composition and behavior are affected in
normal and disease conditions is required for the potential devel-
opment of SG-targeting therapies. A few systematic approaches
have been employed to catalog the protein content of

590 Cell 172, 590–604, January 25, 2018 ª 2017 Elsevier Inc.
SGs (Buchan et al., 2013; Jain et al., 2016; Ohn et al., 2008).
However, these efforts need to be complemented with in vivo
approaches that address potential loss or gain of SG protein
interactions following cell lysis. Furthermore, there is an unmet
need to systematically examine the extent to which SG compo-
sition is dependent on cell type, the nature of the stressor, and
the presence of disease-linked mutations in SG proteins.
In this study, we use a combination of ascorbate peroxidase
(APEX)-mediated in vivo proximity labeling (Rhee et al., 2013)
with quantitative mass spectrometry (MS) and an RBP-focused
immunofluorescence (IF) approach to comprehensively and
significantly expand the repertoire of known SG proteins across
different cell types, stress conditions, and disease states. We
show that SG proteins form a dense protein interaction network
(PIN) in unstressed cells that is poised to enable rapid SG
assembly in response to stress. In addition, we find that SGs in
neuronal cells are particularly diverse in composition and contain
numerous protein quality control (PQC) factors. We reveal aber-
rant composition, behavior, and subcellular localization of SGs
in motor neurons derived from stem cell models harboring
ALS-associated mutations in HNRNPA2B1 and C9orf72. By sys-
tematically integrating our refined SG proteome with published
neurodegeneration-relevant datasets, we provide a framework
for further investigations into the molecular underpinnings of
SG biology and how it relates to human disease. To underscore
the potential of identifying unexpected disease-relevant factors
among SG proteins, we show that known and previously
unknown SG components modify neurotoxicity in Drosophila
models of FUS-, TDP-43-, and C9orf72-mediated degeneration.
We characterize one of these, UBAP2L, as an essential, disor-
dered, and highly aggregation-prone SG protein that can modu-
late ALS phenotypes in vivo.
RESULTS
Endogenously Tagged G3BP1-APEX2-GFP Allows for
Specific Biotin Labeling of SG Proteins
To investigate the protein composition of SGs in living cells, we
performed proximity labeling using an engineered ascorbate
peroxidase (APEX2) fused to the well-characterized SG protein
G3BP1 (Figure 1A). We used CRISPR/Cas9-directed genome
engineering to insert APEX2-GFP into the endogenous G3BP1
locus in HEK293T cells (Figure S1A). The resulting G3BP1-
APEX2-GFP fusion protein allows visualization of SGs upon
sodium arsenite (NaAsO2) exposure, as well as robust and rapid
biotin labeling of SG proteins in the presence of biotin-phenol
(BP) and hydrogen peroxide (H2O2) (Figures 1B and 1C). As a
specificity control, cells with constitutive expression of cyto-
plasmic-localized APEX2 (NES-APEX2-GFP) (Figure S1B) show
a diffuse GFP signal and a biotinylation pattern that is unaffected
by NaAsO2 (Figures 1B and 1C).
Identification of Stress-Dependent and Independent SG
Proteomes Using Quantitative Proteomics
Since G3BP1 is essential for SG formation and robustly localizes
to SGs, we reasoned that defining the interactome proximal to
G3BP1 under stress conditions approximates the SG proteome.
We employed a series of quantitative proteomics experiments
(Figure S1C) to systematically identify three classes of G3BP1-
interacting proteins in stressed and unstressed cells: (1) stress-
independent interactors, which associate with G3BP1 indepen-
dently of stress; (2) stress-dependent partners, which associate
with G3BP1 only under stress; and (3) stress-sensitive interac-
tors, whose association with G3BP1 is lost or weakened during
stress (Figure 1D).
To distinguish these interactors, we pursued four experi-
mental schemes (Figure 1E). First, to identify stress-dependent
G3BP1 interactors, we characterized biotinylated proteins
in stressed versus unstressed G3BP1-APEX2-GFP cells
(experiment 1). Next, we compared lysates from stressed
G3BP1-APEX2-GFP cells incubated with BP to lysates of
identically treated cells for which the BP substrate was
omitted (experiment 2). Third, to control for diffuse cytoplasmic
labeling by G3BP1-APEX2-GFP, we also compared lysates
from stressed G3BP1-APEX2-GFP and NES-APEX2-GFP cells
(experiment 3). Last, to define stress-independent as well as
stress-sensitive G3BP1 interactors, we profiled lysates from
unstressed G3BP1-APEX2-GFP and NES-APEX2-GFP cells
(experiment 4). For each approach, we conducted biologically
independent triplicate labeling reactions followed by mixing of
lysates and streptavidin purification of biotinylated proteins.
Affinity-purified samples and the corresponding input samples
were analyzed by quantitative MS. In total, we detected 1,416
proteins across all input samples and 2,020 proteins across
all streptavidin enrichments (Figure S1D), accounting for 64%
(153) of a manually curated list of 238 annotated SG proteins
(Table S2). Protein identification and quantification of heavy to
light (H/L) ratios were highly reproducible across replicate ex-
periments (Figure S2; Table S1). We compared the enrichment
of known SG proteins to the background distribution of all
detected proteins (Figures 1E and 2A). Known SG proteins
were significantly enriched across all four approaches, with
the greatest shift in log2 H/L ratios detected in experiments
2 and 3. Interestingly, we observed attenuated enrichment of
known SG proteins in experiment 1 and that even in the
absence of stress (experiment 4), known SG proteins appeared
to be enriched in the IP samples (Figures 1E and 2A).
G3BP1-APEX2-Mediated Biotinylation Identifies SG
Proteins with High Specificity
We used a series of analysis steps to identify candidate SG pro-
teins from our quantitative proteomics data (Figure S1E). We first
leveraged our curated list of annotated SG proteins to determine
log2 H/L ratio cutoffs in a non-parametric approach similar to
previous ratiometric SILAC APEX experiments (Hung et al.,
2014). We ranked identified proteins in each replicate by their
log2 H/L ratio and calculated the frequency distribution of known
SG proteins across the ranked lists (Figure 2A). We chose as a
conservative cutoff the ratio at which the frequency of known
SG proteins in a moving window fell below 2-fold the frequency
across all detected proteins. In parallel, we applied an empirical
Bayes method (Kammers et al., 2015) to identify proteins that
were significantly enriched in heavy over light samples. This
method is based on the linear models for microarray data
(LIMMA) approach (Smyth, 2004), which is also applicable to
quantitative proteomics data (Margolin et al., 2009). It uses the
Cell 172, 590–604, January 25, 2018 591
Figure 1. G3BP1-APEX2 Mediates Specific Biotinylation of Stress-Granule-Associated Proteins
(A) Schematic of APEX proximity labeling to tag SG proteins with biotin.
(B) Streptavidin staining of unstressed and NaAsO2-treated HEK293T G3BP1-APEX2-GFP and hPGK-NES-APEX2-GFP cells. Scale bars, 25 mm.
(C) Streptavidin-HRP western blot analysis of induced protein biotinylation in lysates from NES-APEX2-GFP and G3BP1-APEX2-GFP cells.
(D) Schematic of G3BP1 interactome changes upon stress.
(E) Experimental designs for detecting the G3BP1 interactome changes under different conditions, including log2 H/L ratio distributions of all proteins detected,
overlaid with log2 H/L ratio distributions of known SG proteins.
See also Figures S1 and S2 and Table S1.

actual observed data to moderate individual protein variances
through an estimated global sample variance, and thus enables
a more robust identification of significant changes in protein
abundance than ordinary t-statistics. The resulting moderated
p values are corrected for multiple hypothesis testing using
a modified Benjamini-Hochberg false discovery rate (FDR)
approach to determine a moderated q-value (q.mod).
For the final list of SG candidates, we initially selected all
proteins that were above the ratio cutoff in at least 2 out of 3
IP replicates. We defined a set of 123 proteins from the
overlapping sets as shown in Figure 2B (shaded in gray). Of
these, 80% (99/123) are also statistically significantly enriched
(q.mod < 0.05) in at least one experiment. For most of the re-
maining 24 proteins, no significance values could be deter-
mined due to missing data in one of the biological replicates.
However, as 25% of these proteins were previously known
SG proteins, we chose to retain them in our final list (marked
by # in Figure 2B). Table S3 provides a detailed overview of
all hit candidates across all four experimental designs.
Underscoring the robustness of the approach, many well-
characterized SG proteins (e.g., G3BP1, TIA1, CAPRIN1,
PABPC1, FMR1, and ATXN2) were identified as highly significant
interactors across multiple experiments (Figure 2C). In summary,
nearly 80% (96/123) of hits are known SG proteins (69/123), were
verified by IF (13/123), or have additional data supporting SG as-
sociation, such as closely related family members or interactions
with known SG proteins (14/100; e.g., HNRNPDL, YTHDF3). For
example, the DEAD-box helicase DDX1 is known to localize to
SGs and was shown to form an RNA transport complex with
C14ORF166, FAM98A/B, and RTCB (Pérez-González et al.,
2014), all of which we identify as SG candidates (Figures 2B
and 2C). Interestingly, our SG protein set also contains
ANXA11, its closest paralog ANXA7, and their interactor PEF1
(Figures 2B and 2C). While none of these proteins had previously

592 Cell 172, 590–604, January 25, 2018

Figure 2. SG-APEX Identifies Known and Previously Unknown SG Proteins within a Dense Interaction Network
(A) Enrichment frequency distribution of known SG proteins in log2 H/L-ranked proteomics datasets. The dashed line represents 2 times the background
frequency of SG proteins across all detected proteins.
(B) Venn diagram showing overlapping hits from four experimental designs, with previously known SG proteins highlighted in bold.
(C) Volcano plots showing statistically significant enrichment of selected known and previously unknown SG proteins across experiments.
(D) Protein interaction network (PIN) of 283 proteins identified as APEX hits in HEK293T cells or previously shown to associate with SGs. Network was visualized in
Cytoscape using a force-directed layout.
(E) Common network parameters for the SG-PIN compared to five PINs from a randomly selected equal number of nodes and edges.
See also Table S3.

Cell 172, 590–604, January 25, 2018 593

been implicated in SG biology, ANXA11 was recently shown to
harbor ALS-associated mutations leading to abnormal protein
aggregation (Smith et al., 2017). In summary, we demonstrate
that APEX proximity labeling can be applied to dynamic RNP
granules to identify known and previously unknown SG proteins
with relevance to neurodegenerative disease.
Proximity Labeling Reveals a Pre-existing Network of SG
Protein Interactions
Visible SGs only coalesce in response to cellular stress; how-
ever, our data suggested an enrichment of SG proteins in the
G3BP1 interactome even in unstressed cells. Indeed, we found
that less than half (48.8% [60/123]) of SG-APEX hits were
stress-dependent interactors (Figure 2B), including 11 (out of
12 detected) individual subunits of the EIF3 and EIF4 translation
initiation factors, which are thought to accumulate in stalled
pre-initiation complexes in SGs. Remarkably, 51.2% (63/123)
of APEX hits, including many well-studied SG proteins (e.g.,
CAPRIN1, FMR1, TIA1, and USP10), interact with G3BP1 in the
absence of stress (Figures 2B and 2C).
To expand on these findings, we also retrieved publicly avail-
able direct protein-protein interaction (PPI) data for all proteins
either detected by SG-APEX in HEK293T cells or previously
annotated as SG proteins. The resulting SG-PIN contains 283
nodes and 866 non-redundant edges (Figure 2D) and is more
densely connected than PINs built from an equal number of
randomly selected nodes and edges (Figure 2E). In combination
with our SG-APEX data, this suggests a pre-existing steady-
state network of protein interactions that likely facilitates the
rapid coalescence of microscopically visible SGs upon exposure
of cells to environmental stress.
G3BP1-APEX2 Proximity Labeling in Human Neuronal
Cells Reveals Cell-Type- and Stress-Specific SG
Proteins
To enable analysis of SGs in more neurodegeneration-relevant
cell types, we used CRISPR/Cas9-mediated genome engineer-
ing to generate G3BP1-APEX2-GFP-expressing human induced
pluripotent stem cells (iPSCs). Upon differentiation to neural pro-
genitor cells (NPCs) (Figure 3A), G3BP1-APEX2-GFP robustly
localized to SGs upon NaAsO2 treatment and streptavidin stain-
ing overlapped well with the GFP signal (Figure S3A). We then
used quantitative proteomics to compare NaAsO2-treated and
unstressed G3BP1-APEX2-GFP expressing NPCs, as well as
NaAsO2-treated G3BP1-APEX2-GFP cells with and without sub-
strate (Figure S3B). To compare the effects of different stressors,
we used thapsigargin in parallel experiments. In total, we de-
tected 3,880 proteins across all streptavidin enrichments and
input samples that account for 77% (183/238) of known SG pro-
teins (Figure S3C; Table S4). Analysis of log2 H/L ratio distribu-
tions and enrichment of known SG proteins in the data gave
similar results to those observed in HEK293T cells, with high
enrichment of known SG proteins in all experiments (Fig-
ure 3B,C). Using a similar analysis strategy as in HEK293T cells,
we designated 178 proteins from experiments with both
stressors as candidate SG proteins in NPCs (Figure S3D; Table
S4). Of these, 45.5% (81/178) are known SG proteins and
another 21.3% (38/178) were either closely related to known
594 Cell 172, 590–604, January 25, 2018
SG proteins, also identified as SG candidates in HEK293T cells,
or independently validated by IF (Figures 3D and 3E; Table S4).
Interestingly, of the combined 221 SG-APEX hits from NPCs
and HEK293T cells, 64% (141/221) were identified in only one
cell type (Figure 3D). Many of these proteins identified in only
one cell type were not detected in sufficient experiments in the
other cell line, likely due to incomplete capture of lowly ex-
pressed proteins and differences in protein abundance between
cell types. Nevertheless, these findings suggested a potentially
larger than anticipated cell-type-specific diversity of SG compo-
sition and called for further examination through complementary
approaches.
Neuronal SG Proteins Function in Cellular Pathways
Relevant to Neurodegeneration
We next wanted to evaluate whether the seemingly greater
complexity of neuronal SG composition might contribute to
rendering these cell types especially vulnerable to environmental
stress and subsequent protein aggregation. As expected,
several proteins with known neuronal expression and links to
neurodegeneration (e.g., CELF2/3, ELAVL2/3/4, and GIGYF1/2)
were among the neuronal SG proteins. In addition, we validated
the SG association of several previously unknown candidates
involved in the regulation of protein folding (HSPD1, PPIG,
and VBP1) (Figure 3F). A second pillar of proteostasis is clear-
ance of aggregates, and we identified numerous factors that
function in autophagy and related vesicular transport processes
(Figure 3G) among our neuronal SG proteins. Several of these
(GABARAPL2, YLPM1, and SAFB2) cluster in the ULK-AMPK
(AMP-activated protein kinase) subnetwork of the human auto-
phagy system (Behrends et al., 2010), which also contains
constitutive SG proteins (G3BP1, USP10, and CDC37) and
may integrate aging and the cellular stress response (Salminen
and Kaarniranta, 2012). The extent to which SG proteins are sur-
veilled by autophagy factors is further highlighted by the finding
that 5 of the 6 proteins with the highest connectivity to an
extended list of SG candidates comprising all 361 APEX hits
and previously known SG proteins are members of the ATG8
family of small ubiquitin-like modifiers (Figure 3H). Indeed,
16% of all GABARAPL2 (85/539) and 17% of all MAP1LC3A
(65/383) interactions are with SG proteins, which together repre-
sent 2.5% (361/14,352) of all proteins in the PPI dataset (Fig-
ure 3H), suggesting that tight surveillance of SG proteins through
interactions with ATG8 proteins may facilitate the important
role of autophagy in SG clearance (Buchan et al., 2013).
High-Throughput Imaging of RBPs Reveals Stress- and
Cell-Type-Specific SG Composition
As SG proteins are strongly enriched for RBPs, highly validated
antibodies against >300 human RBPs (Sundararaman et al.,
2016) were combined with a screening pipeline involving
systematic IF labeling followed by high-content microscopy
and image analysis (Figure 4A) to further characterize the reper-
toire of SG-associated RBPs. SGs have been known to exhibit
stress-dependent variability in composition (Aulas et al., 2017),
but to our knowledge, the extent of this variability has not been
comprehensively evaluated. To systematically determine the de-
gree to which SG composition varies by stress type, we exposed
Figure 3. NPCs and HEK293T Cells Contain Distinct but Overlapping Sets of SG Proteins
(A) Overview of NPC generation from induced pluripotent stem cells (iPSCs).
(B) Enrichment frequency distribution of known SG proteins in log2 H/L-ranked proteomics datasets. The dashed lines represent 2 times the background
frequency of SG proteins across all detected proteins.
(C) Volcano plot showing statistically significant enrichment of selected known and previously unknown neuronal SG proteins in NPCs.
(D) Venn diagram showing the overlap between known SG proteins and SG-APEX hits identified in HEK293T cells and NPCs.
(E) Previously unknown SG proteins identified by SG-APEX in both HEK293T cells and NPCs.
(F) IF images of selected neuronal SG proteins with functions related to protein folding.
(G) IF images of selected neuronal SG proteins with functions in autophagy and vesicular transport.
(H) Ranked list of proteins with the greatest connectivity to SG proteins as determined by the Enrichr gene enrichment analysis tool.
Scale bars in (A), (F), and (G), 25 mm. See also Figure S3 and Table S4.

HeLa cells to either NaAsO2 or heat shock (30 min at 42 C) and
performed a screen with our RBP antibody collection. Of the 313
RBP antibodies tested, 17% (52 RBPs) localized to SGs. Among
these, 77% (40/52) localized to SGs under both stress condi-
tions, while 23% (12/52) exhibited stress-type-specific SG tar-
geting (Figures 4B–4D; Table S5). For example, UBAP2L robustly
localized to SGs in both stress conditions, while SG-association
of NOLC1 and SF1 was specific to NaAsO2 or heat shock,
respectively (Figures 4B and 4D). We next conducted parallel
screens in three different cell types (HepG2, HeLa, and NPCs)
treated with NaAsO2. We identified a total of 77 SG-RBPs, with
over half of these (42/77) localizing to SGs in all three cell types
and the remaining 35/77 proteins exhibiting varying degrees of
cell-type specificity (Figures 4E–4G; Table S5). For example,
UBAP2L co-localized with SGs in all cell types, while SRSF9,
EIF3A and SRP68 were selectively targeted to SGs in HepG2
cells, HeLa cells, or NPCs, respectively (Figures 4E and 4G).
Notably, consistent with our APEX results, we found that about

Cell 172, 590–604, January 25, 2018 595

Figure 4. An RBP-Centered Imaging Screen Identifies Stress- and Cell-Type-Specific SG Components
(A) High-content imaging (HCI) screen outline to identify SG-localized RBPs in HepG2 cells, HeLa cells, and NPCs.
(B) IF images showing examples of RBP localization in untreated, NaAsO2 (AS)-treated, and heat-shock (HS)-treated HeLa cells. UBAP2L is a common hit in both
stress conditions; NOLC1 and SF1 are specific to NaAsO2 and heat shock, respectively. Left panels are merged lower-resolution views, and right panels
represent zoomed-in views of the indicated selection separately showing TIA-1 (red) or the test RBP (green). Arrowheads indicate co-localization of the test
RBP with TIA1.
(C) Venn diagram comparing SG proteins in HeLa cells treated with NaAsO2 versus heat shock.
(D) Quantification of the mean granule penetrance of proteins with either constitutive (UBAP2L) or stress-type-specific (NOLC1 and SF1) SG localization.
(E) IF images showing examples of RBP localization in untreated and NaAsO2-treated HeLa cells, HepG2 cells, or NPCs. UBAP2L is found in SGs in all three cell
types, while SRSF9, EIF3A, and SRP68 are specific to HepG2 cells, HeLa cells, and NPCs, respectively. Top panels are merged lower-resolution views, while the
(legend continued on next page)

596 Cell 172, 590–604, January 25, 2018

one third (28/77) of SG-RBPs localized to SGs in NPCs but failed
to do so in at least one of the other cell types tested. To summa-
rize, 120 proteins were found to associate with SGs in NPCs but
were absent from SGs in at least one other cell type. While these
proteins may not be exclusive to SGs in neuronal cells, we refer
to them as neuronal SG proteins here because they show a
neuronal preference due to either higher expression levels or
cell-type-specific SG targeting. This systematic survey of cell-
type-specific SG-RBPs further extends our SG compendium
and shows that neuronal cells exhibit a greater diversity in SG
composition than non-neuronal cells. The observation that
75% (90/120) of neuronal SG proteins had not previously been
associated with SGs further highlights that past studies using
common immortalized cell lines have missed potentially impor-
tant neuronal SG proteins.
SGs Vary in Composition and Subcellular Localization in
iPSC-Derived Motor Neurons
The components of SGs, as well as the molecular interactions
that determine SG dynamics are increasingly implicated in
human neurological disorders, including ALS. As motor neurons
(MNs) are the most severely affected cell type in ALS, we next
characterized the SG-targeting behavior of RBPs in iPSC-
derived MNs (iPS-MNs; Figure S4A). We first carried out IF stain-
ing for 63 (of 77) SG-RBP hits in control iPS-MNs that were either
untreated or treated with NaAsO2 or puromycin, which robustly
induces SGs in iPS-MNs after a 24-hr treatment without overt
toxicity (Martinez et al., 2016). In unstressed iPS-MNs, 57%
(36/63) of RBPs localized primarily to cell bodies (e.g., IGF2BP3;
Figures 5A and 5B), whereas 43% (27/63) of RBPs also showed
clear localization to projecting neurites (e.g., SND1; Figures 5A
and 5B). Following stress treatments, we identified 51 RBPs
that co-localized with G3BP1-labeled SGs (Table S6), most of
which (49/51) localized to SGs in both NaAsO2- or puromycin-
treated cells, while two (DAZAP1, ZC3H11A) were selectively tar-
geted in response to puromycin. Both SND1 and IGF2BP3 co-
localized with G3BP1-labeled SGs in cell bodies, while SND1
was also present in granules along neurites (Figure 5A). We
conclude that stress-induced granules of varying composition
form in a subcellular compartment-specific manner in human
iPS-MNs.
SG Composition and Subcellular Distribution Are
Affected in iPSC Models of ALS
ALS-associated mutant versions of FUS, hnRNPA2/B1, and
TIA1, as well as dipeptide repeats (DPRs) derived from an
expanded GGGGCC (G4C2) repeat in C9orf72, were recently
reported to affect rate and dynamics of SG formation
(Boeynaems et al., 2017; Lee et al., 2016; Lin et al., 2016;
Mackenzie et al., 2017; Martinez et al., 2016; Murakami et al.,
2015; Patel et al., 2015). Expanding on our previous study
using iPS-MNs carrying the ALS-associated D290V mutation
bottom panels represent zoomed-in views of the indicated selection separately showing TIA-1 (red) or the test RBP (green). Arrowheads indicate examples
of RBPs co-localized with TIA-1.
(F) Venn diagram comparing SG proteins in HepG2, HeLa and NPCs treated with NaAsO2.
(G) Mean granule penetrance of proteins with either cell-type-independent or cell-type-specific SG localization.
Scale bars in (B) and (E), 20 mm. Error bars in (D) and (G) represent SD. See also Table S5.
in HNRNPA2B1 (Martinez et al., 2016), we observed an
increased rate of formation as well as impaired early
clearance of puromycin-induced G3BP1-positive SGs in both
HNRNPA2B1 and C9orf72 mutant iPS-MNs (Figure 5C).
Surprisingly, in addition to an increased propensity to form
SGs (Figure 5E), screening of HNRNPA2B1 mutant iPS-MNs
with our SG-RBP antibodies also revealed mutation-specific
differences in the subcellular distribution of SG-RBPs (Figures
5D and 5F). In control cells, most analyzed RBPs localize
primarily to SGs in the cell body upon puromycin treatment.
In contrast, almost half (23/50) of SG-RBPs also localized to
prominent granules in neurites in HNRNPA2B1 mutant cells
(such as IGF2BP1/2/3, SAFB2, PCBP2, NKRF, and FAM120A;
shown in Figures 5D and 5F; Figure S4B). Additionally, even
for proteins that were found in neurite-localized granules in
both control and mutant cells (such as the ALS-associated
protein TDP-43), their localization to neurite-localized SGs
appeared more pronounced in HNRNPA2B1 mutant cells
(Figure 5D). The RBPs found in neurite-localized SGs were
enriched in functions such as RNA transport and translational
suppression (Figure S4C), which is consistent with and extends
previous studies reporting that axonal transport is impaired in
ALS motor neurons (Alami et al., 2014; Yasuda and Mili,
2016). Altogether, our findings confirm aberrant SG dynamics
across ALS-associated mutations and highlight abnormal SG
distribution and composition in the projections of HNRNPA2B1
mutant iPS-MNs, which provide further insights into ALS
pathogenesis.
Cross-Comparison with Related Datasets Identifies
Disease-Relevant SG Proteins
Together, our APEX and IF screening approaches identified 260
SG proteins, including 150 candidates that had not previously
been associated with SGs. Consistent with known SG proteins,
our hits are enriched for RBPs (201/260 [77.3%]; Figure 6A) with
a range of RNA-binding domains (Figure 6B) and gene ontology
(GO) terms associated with RNA metabolism and translational
control (Figure 6C). They also contain a significantly higher pro-
portion of amino acid residues in IDRs and LCDs (Figure 6D)
than the background proteome, consistent with LLPS being a
driver of SG assembly. To place in context how individual SG
proteins might be connected to disease, we integrated our SG
compendium with 11 published datasets related to protein aggre-
gation in neurodegeneration (Figure 6E; Table S7) (Blokhuis et al.,
2016; Freibaum et al., 2010; Jain et al., 2016; Kato et al., 2012; Lee
et al., 2016; Lin et al., 2016; March et al., 2016). While these
diverse datasets are not expected to overlap completely, their
cross-comparison can nevertheless be useful for situating each
individual study into a greater context. Furthermore, ranking the
proteins by how frequently they occur across all datasets can
help identify features of the most consistently observed proteins
and prioritize candidate genes for follow-up studies.
Cell 172, 590–604, January 25, 2018 597
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598 Cell 172, 590–604, January 25, 2018

 Of the 1,312 proteins found across the 14 datasets, almost two
thirds (840/1,312) were present in only a single dataset (Fig-
ure S5B) and only 5% (71/1,312) were found in at least half of
the datasets. Remarkably, all of these 71 proteins and indeed
96% (192/200) of the top 200 proteins are RBPs, many with
higher proportions of LCDs and IDRs (Figure 6E) than the back-
ground proteome. Many well-studied SG proteins with roles in
neurodegeneration (e.g., FUS, ATXN2, and FMR1) are broadly
represented, as are several less well-characterized and previ-
ously unknown SG proteins that warrant further investigation.
SG Components Modify Disease Protein Toxicity in
Drosophila ALS/FTD Models
To further confirm the disease-relevance of known and previ-
ously unidentified SG proteins in vivo, we used several
Drosophila models of ALS/FTD to examine disease protein
toxicity in both neuronal and non-neuronal cells. Mis-expression
of ALS-linked mutated hTDP-43M337V or hFUSR521C, respec-
tively, causes a neurodegenerative rough eye phenotype
(unpublished data; Lanson et al., 2011; Ritson et al., 2010). We
recently carried out a genome-wide screen for genetic modifiers
of TDP-43 and FUS toxicity (unpublished data), which we inter-
sected with our SG protein data. Not unexpectedly, we identified
several SG-RBPs as modifiers of TDP-43 and FUS-mediated
toxicity (Figure 6F). In addition to the TDP-43 and FUS models,
we performed genetic interaction studies to test several selected
SG proteins for their ability to modify toxicity caused by overex-
pression of a C9orf72-ALS/FTD associated poly(GR) in the
Drosophila wing (Yang et al., 2015). We tested 9 candidate
genes, either by multiple RNAi knockdown or through genetic
mutant alleles, and found that partial loss of activity for more
than half (5/9) significantly rescued poly(GR) toxicity (Figure 6G;
Figure S5C). Although the detailed mechanisms underlying
these genetic interactions remain to be further investigated,
our Drosophila results identify several previously unknown SG
proteins, such as CBX3, CSDE1, RBMS1/2, UBAP2(L), and
YEATS2, as potentially disease-relevant factors (Figures 6F
and 6G), underscoring again that alterations in SG components
can affect neurodegenerative phenotypes.
We selected one of these previously unknown SG-associ-
ated disease-modifiers, UBAP2L, for evaluation in human cells,
as it was consistently among the most robustly SG-associated
proteins by both SG-APEX and IF across all cell types and
stress conditions tested (Figures 4B and 4E). We found that
depletion of UBAP2L by small interfering RNA (siRNA) in
HeLa cells almost completely abolished NaAsO2-induced SG
formation (Figure 6H), establishing UBAP2L as an essential
regulator of SG assembly. It is among the most disordered pro-
teins in the human proteome, with 99.4% of its 1,087 amino
acids considered to fall within IDRs (Figure S5D), suggesting
a potential for UBAP2L to undergo LLPS. Interestingly, while
inducible expression of an UBAP2L-mCherry fusion protein
recapitulated endogenous UBAP2L localization, a truncated
version lacking the N-terminal ubiquitin-associated UBA
domain (DUBA_UBAP2L-mCherry) led to widespread formation
of aggregates containing the SG proteins G3BP1, FMR1, and
ELAVL1 even in the absence of stress (Figure 6I; Figure S5E).
Our findings reveal UBAP2L as an excellent future candidate
to study how protein aggregation might be regulated both in
the absence or presence of cellular stress.
DISCUSSION
In this study, we extend the application of in vivo APEX proximity
labeling combined with quantitative proteomics to the study of
highly dynamic, non-membranous RNP granules. In combination
with an RBP-focused IF screen, we identify 150 proteins not
previously known to associate with SGs. We estimate that up
to 20% of components may be recruited to SGs in a cell-type-
or stress-type-specific manner. Interestingly, neuronal cells
appear to contain compositionally more diverse SGs, and
many of the components have reported functions in PQC path-
ways such as chaperone-assisted protein folding and aggregate
clearance by autophagy. Defects in PQC have been implicated in
the development of neurodegeneration (Ciechanover and Kwon,
2017), and our observation might help explain why neuronal
cells are especially dependent on accurate regulation of protein
homeostasis. We implemented our SG-APEX approach by
tagging the endogenous G3BP1 locus in iPSCs, which not only
minimizes potential artifacts from G3BP-APEX2-GFP overex-
pression but also opens the possibility of studying SGs in a
wide range of differentiated cell types from a constant genetic
background. Future studies using a combination of different
APEX2-tagged proteins will make it possible to further dissect
the molecular architecture of RNPs and enable the distinction
of closely related subtypes of RNP granules such as P-bodies,
as well as the characterization of cell-type-specific granules
such as neuronal transport granules.

Figure 5. SG Composition and Subcellular Distribution Is Altered in ALS-Patient-Derived iPS-MNs

(A) IF images of SND1 and IGF2BP3 localization in unstressed or NaAsO2-treated iPS-MNs. Top panels are merged lower-resolution views, while the
bottom panels represent zoomed-in views of the indicated selection separately showing G3BP1 (green) or the test RBP (red). Arrowheads indicate examples of
RBPs co-localized with G3BP1.
(B) Overview of RBPs whose localization in unstressed iPS-MNs is either restricted to the cell body or extends into neuronal projections.
(C) Time-course analysis of SG formation in iPS-MNs from controls or from ALS patients bearing mutations in HNRNPA2B1 or C9orf72, respectively.
(D) IF images of control and HNRNPA2B1 mutant iPS-MNs that were either untreated or stressed with puromycin. Top panels are merged lower-resolution views,
while the bottom panels represent zoomed-in views of the indicated selection separately showing G3BP1 (green) or the test RBP (red). White and yellow
arrowheads indicate examples of SGs formed in cell bodies or neurites, respectively.
(E) Quantification of SG area and number in untreated or stressed control and HNRNPA2B1 mutant iPS-MNs.
(F) Quantification of RBPs that localize to SGs in cell bodies or dendritic projections in control versus HNRNPA2B1 mutant cells. The RBPs exhibiting targeting to
SGs in projections in HNRNPA2B1 mutant cells are highlighted in the panel on the right.
Scale bars, 20 mm. Error bars in (C) and (E) represent SD. Statistical significance was calculated by 2-way ANOVA (C) or Student’s t test (E). *p % 0.05; **p % 0.01;
***p % 0.001; ****p % 0.0001. See also Figure S4 and Table S6.

Cell 172, 590–604, January 25, 2018 599

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600 Cell 172, 590–604, January 25, 2018

Figure 7. SG Form from Pre-existing PPIs Are Especially Diverse in Neuronal Cells and Display Aberrant Characteristics in ALS Mutant Cells
(A) Model of the relationships among normally functioning, dynamic RNPs, transient SGs, and permanent pathological protein inclusions.
(B) Schematic showing that neuronal SGs are diverse and contain proteins involved in protein quality control pathways.
(C) Schematic showing altered SG dynamics, composition, and subcellular distribution in ALS mutant motor neurons.

In addition to providing a resource of nearly 150 previously
unknown candidate SG proteins for further validation, our study
links many known and previously unidentified SG proteins to
human disease and provides unexpected and exciting insights
into SG biology and how it relates to neurodegeneration (Fig-
ure 7). First, our SG-APEX data in stressed and unstressed cells,
combined with independent PPI data, show that much of the
underlying network of SG protein interactions already exists in
unstressed cells. This finding sharpens the picture of a highly
evolved and dense network of RNPs that integrates the many
steps of gene expression regulation. As a result, although SGs
appear to form de novo in response to stress, their emergence
represents a moderate and transient shift in a tightly controlled
equilibrium of protein-protein and protein-RNA interactions
(Figure 7A). Allocating high local concentrations of processing
factors and substrates into interconnected RNP assemblies
enables highly efficient processing to take place but at the
same time increases the risk of uncontrolled protein aggregation.
As a result, cells have evolved mechanisms for efficiently
resolving transient higher-order RNP assemblies, especially in
the context of a temporary stress response. Our results highlight
how SG proteins are tightly integrated with PQC pathways, most

Figure 6. Integrative Data Analysis Highlights Potential Disease-Relevant Proteins

(A) Venn diagram showing overlap between proteins identified in our combined APEX-IF approach, known SG proteins, and RBPs.
(B) Protein domain enrichment analysis of 260 SG APEX-IF.
(C) Gene ontology analysis for 260 APEX-IF hits.
(D) Comparison of the proportion of amino acids in LCDs and IDRs between APEX-IF hits and background.
(E) Heatmap for the 75 proteins most broadly represented across selected SG and neurodegeneration-relevant datasets. Heatmap indicates whether a protein
is present (blue box) or absent (white box) from each dataset, and proteins are ranked by the number of datasets they are part of in descending order from
left to right.
(F) Images of Drosophila eye degeneration models crossed with the indicated strains.
(G) Images and quantitation of the wing notching phenotype caused by poly(GR) toxicity in flies. w1118 flies were used as the control for genetic mutant alleles,
while UAS-GFP served as the control for different UAS-RNAi lines. Numbers indicate Bloomington stock numbers for each mutant or RNAi line.
(H) IF images of G3BP1 staining and quantification of SG numbers in HeLa cells treated with control siRNA or siRNA targeting UBAP2L. Data are presented as
mean ± SEM, and statistical significance was determined by two-tailed unpaired t test.
(I) IF images of 293FITR cells with inducible expression of either a full-length UBAP2L-mCherry fusion protein (top panel) or a truncated UBAP2L-mCherry fusion
protein missing the N-terminal UBA domain (middle and bottom panels).
Scale bars in (H) are 25 mm. See also Figure S5 and Table S7.

Cell 172, 590–604, January 25, 2018 601

strikingly through close surveillance by the ATG8 family of auto-
phagy mediators. Interestingly, neuronal cells display a greater
diversity in SG composition compared to non-neuronal cells,
and numerous PQC factors localize specifically to neuronal
SGs, potentially providing an explanation to why neurons are
especially vulnerable to environmental stresses (Figure 7B).
Lastly, we demonstrate that iPS-MNs harboring ALS-associated
mutations in HNRNPA2B1 form SGs more readily and that
mutant cells are more prone to forming SGs along neuronal pro-
jections, which differ in their composition from SGs found in the
soma (Figure 7C). These neurite-localized SGs are enriched in
proteins involved in RNA transport and translational repression,
suggesting a mechanism by which genetic mutations could
interact with environmental factors to widely impair axonal trans-
port and contribute to axon degeneration in ALS.
Much attention is currently being focused on understanding
how exactly the known ALS-linked mutations in multifunctional
RBPs alter the structure and function of these proteins to result
in aberrant protein aggregation. However, as emphasized by
the late onset and cell-type specificity of disease symptoms,
these genetic factors only result in pathology once other compo-
nents of the regulatory system that normally prevent long-lived
RNP aggregation begin to fail. Future work should focus on iden-
tifying the critical factors and mechanisms in this system. The SG
protein compendium we present here suggests possible future
directions and provides a framework for identifying previously
unknown important regulators. We present the example of
UBAP2L, which is not only essential for SG formation but also
can seed spontaneous protein aggregates when the ubiquitin-
associated region of the protein is removed. It is likely that mod-
ulation of UBAP2L levels can have a similar effect as reduction of
ATXN2, which was recently shown to ameliorate TDP-43 toxicity
in mice (Becker et al., 2017). Beyond UBAP2L, many poorly char-
acterized proteins with potential relevance to aggregation can be
identified and prioritized from our cross-comparison of more
than a dozen SG- and neurodegeneration-associated PPI data-
sets, combined with the added biological context of whether a
specific RBP localizes to aberrant SG in ALS mutant iPS-MNs
or can modify neurodegenerative phenotypes in flies.
In summary, it is critical to recognize that broad inhibition of
the PPIs that underlie SG formation will also affect the dense
RNP network in unstressed cells. Promising therapeutic strate-
gies will therefore likely need to specifically target those mecha-
nisms that only result in aberrant insoluble protein inclusions.
Our work represents a step along this path, which so far has
been hindered by a sparsity of promising targets as well as a
lack of robust disease-associated phenotypes in living cells.
STAR+METHODS
Detailed methods are provided in the online version of this paper
and include the following:
d KEY RESOURCES TABLE
d CONTACT FOR REAGENT AND RESOURCE SHARING
d EXPERIMENTAL MODEL AND SUBJECT DETAILS
d METHOD DETAILS
B Generation and maintenance of neural progenitor cells
602 Cell 172, 590–604, January 25, 2018
B Generation of iPSC-derived motor neurons
B Plasmid construction
B SILAC labeling with isotopically modified amino acids
B APEX-mediated biotinylation
B IF, imaging and image analysis
B Drosophila genetics
B Protein interaction network analysis
B Protein domain and gene ontology analysis
d QUANTIFICATION AND STATISTICAL ANALYSES
B MS data collection and analysis
B Image analysis
d DATA AND SOFTWARE AVAILABILITY
SUPPLEMENTAL INFORMATION
Supplemental Information includes five figures, eight tables, and one dataset
and can be found with this article online at https://doi.org/10.1016/j.cell.
2017.12.032.
ACKNOWLEDGMENTS
We acknowledge members of the G.W.Y. lab for critical comments. We thank
Shashank Sathe for help with bioinformatics analyses and K.G. Guruharsha
and Maria D. Lalioti for valuable comments on the manuscript. S.M. was sup-
ported by a postdoctoral fellowship from the Larry L. Hillblom Foundation
(2014-A-027-FEL). S.S. was supported by the IRCM Angelo Pizzagalli fellow-
ship. J.M.W. was supported by an NIH Graduate Training in Cellular and
Molecular Pharmacology Training Grant T32 GM007752. G.W.Y. is an Alfred
P. Sloan Research Fellow, and E.L. is an FRQS Junior 2 Scholar. Work in the
Lécuyer lab was supported by an ALS Canada/Brain Canada Discovery Grant.
Work in the Gao lab was supported by the NIH (R01NS101986), the Packard
Center for ALS Research, and the Target ALS Foundation. Work in the Bennett
lab was supported by a Hellman Fellowship and the NIH (DP2GM119132). This
work was partially supported by grants from the NIH (NS103172 and
HG004659) and the ALS Association (to G.W.Y.).
AUTHOR CONTRIBUTIONS
Conceptualization, S.M. and G.W.Y; Methodology, S.M., E.J.B., E.L., and
G.W.Y.; Formal Analysis, S.M., W.J., A.F., J.M.W., and E.J.B.; Investigation,
S.M., S.S., K.L.S., R.M., M.Y.F., E.-C.L., E.L., F.K., D.Y., A.S., M.W.K. and
F.-B.G.; Writing – Original Draft, S.M.; Writing – Review & Editing, S.M.,
E.J.B., E.L., and G.W.Y.; Visualization, S.M. and S.S.; Supervision, D.J.G.,
F.-B.G., E.J.B., E.L., and G.W.Y.; Funding Acquisition, E.J.B., E.L., and G.W.Y.
DECLARATION OF INTERESTS
G.W.Y. is a co-founder of Locana and Eclipse Bioinnovations and member of
the scientific advisory boards of Locana, Eclipse Bioinnovations and Aquinnah
Pharmaceuticals. The terms of this arrangement have been reviewed and
approved by the University of California, San Diego in accordance with its
conflict of interest policies. A.S. and M.W.K. are employees of Biogen. All other
authors declare no competing interests.
Received: June 19, 2017
Revised: October 4, 2017
Accepted: December 19, 2017
Published: January 25, 2018
REFERENCES
Ainger, K., Avossa, D., Morgan, F., Hill, S.J., Barry, C., Barbarese, E., and
Carson, J.H. (1993). Transport and localization of exogenous myelin basic
protein mRNA microinjected into oligodendrocytes. J. Cell Biol. 123,
431–441.
Alami, N.H., Smith, R.B., Carrasco, M.A., Williams, L.A., Winborn, C.S., Han,
S.S.W., Kiskinis, E., Winborn, B., Freibaum, B.D., Kanagaraj, A., et al. (2014).
Axonal transport of TDP-43 mRNA granules is impaired by ALS-causing muta-
tions. Neuron 81, 536–543.
Aulas, A., Fay, M.M., Lyons, S.M., Achorn, C.A., Kedersha, N., Anderson, P.,
and Ivanov, P. (2017). Stress-specific differences in assembly and composi-
tion of stress granules and related foci. J. Cell Sci. 130, 927–937.
Becker, L.A., Huang, B., Bieri, G., Ma, R., Knowles, D.A., Jafar-Nejad, P.,
Messing, J., Kim, H.J., Soriano, A., Auburger, G., et al. (2017). Therapeutic
reduction of ataxin-2 extends lifespan and reduces pathology in TDP-43
mice. Nature 544, 367–371.
Beckmann, B.M., Horos, R., Fischer, B., Castello, A., Eichelbaum, K.,
Alleaume, A.M., Schwarzl, T., Curk, T., Foehr, S., Huber, W., et al. (2015).
The RNA-binding proteomes from yeast to man harbour conserved
enigmRBPs. Nat. Commun. 6, 10127.
Behrends, C., Sowa, M.E., Gygi, S.P., and Harper, J.W. (2010). Network orga-
nization of the human autophagy system. Nature 466, 68–76.
Blokhuis, A.M., Koppers, M., Groen, E.J., van den Heuvel, D.M., Dini Modi-
gliani, S., Anink, J.J., Fumoto, K., van Diggelen, F., Snelting, A., Sodaar, P.,
et al. (2016). Comparative interactomics analysis of different ALS-associated
proteins identifies converging molecular pathways. Acta Neuropathol. 132,
175–196.
Boeynaems, S., Bogaert, E., Kovacs, D., Konijnenberg, A., Timmerman, E.,
Volkov, A., Guharoy, M., De Decker, M., Jaspers, T., Ryan, V.H., et al.
(2017). Phase separation of C9orf72 dipeptide repeats perturbs stress granule
dynamics. Mol. Cell 65, 1044–1055.
Brannan, K.W., Jin, W., Huelga, S.C., Banks, C.A., Gilmore, J.M., Florens, L.,
Washburn, M.P., Van Nostrand, E.L., Pratt, G.A., Schwinn, M.K., et al. (2016).
SONAR discovers RNA-binding proteins from analysis of large-scale protein-
protein interactomes. Mol. Cell 64, 282–293.
Buchan, J.R., Kolaitis, R.M., Taylor, J.P., and Parker, R. (2013). Eukaryotic
stress granules are cleared by autophagy and Cdc48/VCP function. Cell
153, 1461–1474.
Ciechanover, A., and Kwon, Y.T. (2017). Protein quality control by molecular
chaperones in neurodegeneration. Front. Neurosci. 11, 185.
Conrad, T., Albrecht, A.S., de Melo Costa, V.R., Sauer, S., Meierhofer, D., and
Ørom, U.A. (2016). Serial interactome capture of the human cell nucleus.
Nat. Commun. 7, 11212.
Cox, J., and Mann, M. (2008). MaxQuant enables high peptide identification
rates, individualized p.p.b.-range mass accuracies and proteome-wide
protein quantification. Nat. Biotechnol. 26, 1367–1372.
Du, Z.W., Chen, H., Liu, H., Lu, J., Qian, K., Huang, C.L., Zhong, X., Fan, F., and
Zhang, S.C. (2015). Generation and expansion of highly pure motor neuron
progenitors from human pluripotent stem cells. Nat. Commun. 6, 6626.
Dull, T., Zufferey, R., Kelly, M., Mandel, R.J., Nguyen, M., Trono, D., and
Naldini, L. (1998). A third-generation lentivirus vector with a conditional
packaging system. J. Virol. 72, 8463–8471.
Freibaum, B.D., Chitta, R.K., High, A.A., and Taylor, J.P. (2010). Global
analysis of TDP-43 interacting proteins reveals strong association with RNA
splicing and translation machinery. J. Proteome Res. 9, 1104–1120.
Gendron, J.M., Webb, K., Yang, B., Rising, L., Zuzow, N., and Bennett, E.J.
(2016). Using the ubiquitin-modified proteome to monitor distinct and spatially
restricted protein homeostasis dysfunction. Mol. Cell. Proteomics 15,
2576–2593.
Gore, A., Li, Z., Fung, H.L., Young, J.E., Agarwal, S., Antosiewicz-Bourget, J.,
Canto, I., Giorgetti, A., Israel, M.A., Kiskinis, E., et al. (2011). Somatic coding
mutations in human induced pluripotent stem cells. Nature 471, 63–67.
Hung, V., Zou, P., Rhee, H.W., Udeshi, N.D., Cracan, V., Svinkina, T., Carr,
S.A., Mootha, V.K., and Ting, A.Y. (2014). Proteomic mapping of the human
mitochondrial intermembrane space in live cells via ratiometric APEX tagging.
Mol. Cell 55, 332–341.
Jain, S., Wheeler, J.R., Walters, R.W., Agrawal, A., Barsic, A., and Parker, R.
(2016). ATPase-modulated stress granules contain a diverse proteome and
substructure. Cell 164, 487–498.
Kammers, K., Cole, R.N., Tiengwe, C., and Ruczinski, I. (2015). Detecting
significant changes in protein abundance. EuPA Open Proteom. 7, 11–19.
Kato, M., Han, T.W., Xie, S., Shi, K., Du, X., Wu, L.C., Mirzaei, H., Goldsmith,
E.J., Longgood, J., Pei, J., et al. (2012). Cell-free formation of RNA granules:
low complexity sequence domains form dynamic fibers within hydrogels.
Cell 149, 753–767.
Kedersha, N., and Anderson, P. (2002). Stress granules: sites of mRNA triage
that regulate mRNA stability and translatability. Biochem. Soc. Trans. 30,
963–969.
Kedersha, N.L., Gupta, M., Li, W., Miller, I., and Anderson, P. (1999). RNA-
binding proteins TIA-1 and TIAR link the phosphorylation of eIF-2 alpha to
the assembly of mammalian stress granules. J. Cell Biol. 147, 1431–1442.
Kuleshov, M.V., Jones, M.R., Rouillard, A.D., Fernandez, N.F., Duan, Q.,
Wang, Z., Koplev, S., Jenkins, S.L., Jagodnik, K.M., Lachmann, A., et al.
(2016). Enrichr: a comprehensive gene set enrichment analysis web server
2016 update. Nucleic Acids Res. 44 (W1), W90-7.
Lagier-Tourenne, C., Baughn, M., Rigo, F., Sun, S., Liu, P., Li, H.R., Jiang, J.,
Watt, A.T., Chun, S., Katz, M., et al. (2013). Targeted degradation of sense and
antisense C9orf72 RNA foci as therapy for ALS and frontotemporal degenera-
tion. Proc. Natl. Acad. Sci. USA 110, E4530–E4539.
Lam, S.S., Martell, J.D., Kamer, K.J., Deerinck, T.J., Ellisman, M.H., Mootha,
V.K., and Ting, A.Y. (2015). Directed evolution of APEX2 for electron micro-
scopy and proximity labeling. Nat. Methods 12, 51–54.
Lanson, N.A., Jr., Maltare, A., King, H., Smith, R., Kim, J.H., Taylor, J.P., Lloyd,
T.E., and Pandey, U.B. (2011). A Drosophila model of FUS-related neurode-
generation reveals genetic interaction between FUS and TDP-43. Hum. Mol.
Genet. 20, 2510–2523.
Lee, K.H., Zhang, P., Kim, H.J., Mitrea, D.M., Sarkar, M., Freibaum, B.D., Cika,
J., Coughlin, M., Messing, J., Molliex, A., et al. (2016). C9orf72 dipeptide
repeats impair the assembly, dynamics, and function of membrane-less
organelles. Cell 167, 774–788.
Lin, Y., Mori, E., Kato, M., Xiang, S., Wu, L., Kwon, I., and McKnight, S.L.
(2016). Toxic PR poly-dipeptides encoded by the C9orf72 repeat expansion
target LC domain polymers. Cell 167, 789–802.
Mackenzie, I.R., Nicholson, A.M., Sarkar, M., Messing, J., Purice, M.D.,
Pottier, C., Annu, K., Baker, M., Perkerson, R.B., Kurti, A., et al. (2017). TIA1
mutations in amyotrophic lateral sclerosis and frontotemporal dementia
promote phase separation and alter stress granule dynamics. Neuron 95,
808–816.
March, Z.M., King, O.D., and Shorter, J. (2016). Prion-like domains as epige-
netic regulators, scaffolds for subcellular organization, and drivers of neurode-
generative disease. Brain Res. 1647, 9–18.
Margolin, A.A., Ong, S.E., Schenone, M., Gould, R., Schreiber, S.L., Carr, S.A.,
and Golub, T.R. (2009). Empirical Bayes analysis of quantitative proteomics
experiments. PLoS ONE 4, e7454.
Martinez, F.J., Pratt, G.A., Van Nostrand, E.L., Batra, R., Huelga, S.C., Kapeli,
K., Freese, P., Chun, S.J., Ling, K., Gelboin-Burkhart, C., et al. (2016). Protein-
RNA networks regulated by normal and ALS-associated mutant HNRNPA2B1
in the nervous system. Neuron 92, 780–795.
Molliex, A., Temirov, J., Lee, J., Coughlin, M., Kanagaraj, A.P., Kim, H.J.,
Mittag, T., and Taylor, J.P. (2015). Phase separation by low complexity
domains promotes stress granule assembly and drives pathological fibrilliza-
tion. Cell 163, 123–133.
Murakami, T., Qamar, S., Lin, J.Q., Schierle, G.S., Rees, E., Miyashita, A.,
Costa, A.R., Dodd, R.B., Chan, F.T., Michel, C.H., et al. (2015). ALS/FTD
mutation-induced phase transition of FUS liquid droplets and reversible
hydrogels into irreversible hydrogels impairs RNP granule function. Neuron
88, 678–690.
Cell 172, 590–604, January 25, 2018 603
Ohn, T., Kedersha, N., Hickman, T., Tisdale, S., and Anderson, P. (2008). A
functional RNAi screen links O-GlcNAc modification of ribosomal proteins to
stress granule and processing body assembly. Nat. Cell Biol. 10, 1224–1231.
Patel, A., Lee, H.O., Jawerth, L., Maharana, S., Jahnel, M., Hein, M.Y.,
Stoynov, S., Mahamid, J., Saha, S., Franzmann, T.M., et al. (2015). A liquid-
to-solid phase transition of the ALS protein FUS accelerated by disease
mutation. Cell 162, 1066–1077.
Pérez-González, A., Pazo, A., Navajas, R., Ciordia, S., Rodriguez-Frandsen,
A., and Nieto, A. (2014). hCLE/C14orf166 associates with DDX1-HSPC117-
FAM98B in a novel transcription-dependent shuttling RNA-transporting
complex. PLoS ONE 9, e90957.
Periz, G., Lu, J., Zhang, T., Kankel, M.W., Jablonski, A.M., Kalb, R.,
McCampbell, A., and Wang, J. (2015). Regulation of protein quality control
by UBE4B and LSD1 through p53-mediated transcription. PLoS Biol. 13,
e1002114.
Ran, F.A., Hsu, P.D., Wright, J., Agarwala, V., Scott, D.A., and Zhang, F. (2013).
Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 8,
2281–2308.
Reinhardt, P., Glatza, M., Hemmer, K., Tsytsyura, Y., Thiel, C.S., Höing, S.,
Moritz, S., Parga, J.A., Wagner, L., Bruder, J.M., et al. (2013). Derivation and
expansion using only small molecules of human neural progenitors for neuro-
degenerative disease modeling. PLoS ONE 8, e59252.
Rhee, H.W., Zou, P., Udeshi, N.D., Martell, J.D., Mootha, V.K., Carr, S.A., and
Ting, A.Y. (2013). Proteomic mapping of mitochondria in living cells via
spatially restricted enzymatic tagging. Science 339, 1328–1331.
Ritson, G.P., Custer, S.K., Freibaum, B.D., Guinto, J.B., Geffel, D., Moore, J.,
Tang, W., Winton, M.J., Neumann, M., Trojanowski, J.Q., et al. (2010). TDP-43
mediates degeneration in a novel Drosophila model of disease caused by
mutations in VCP/p97. J. Neurosci. 30, 7729–7739.
604 Cell 172, 590–604, January 25, 2018
Salminen, A., and Kaarniranta, K. (2012). AMP-activated protein kinase
(AMPK) controls the aging process via an integrated signaling network. Ageing
Res. Rev. 11, 230–241.
Shannon, P., Markiel, A., Ozier, O., Baliga, N.S., Wang, J.T., Ramage, D., Amin,
N., Schwikowski, B., and Ideker, T. (2003). Cytoscape: a software environment
for integrated models of biomolecular interaction networks. Genome Res. 13,
2498–2504.
Smith, B.N., Topp, S.D., Fallini, C., Shibata, H., Chen, H.J., Troakes, C., King,
A., Ticozzi, N., Kenna, K.P., Soragia-Gkazi, A., et al. (2017). Mutations in the
vesicular trafficking protein annexin A11 are associated with amyotrophic
lateral sclerosis. Sci. Transl. Med. 9, eaad9157.
Smyth, G.K. (2004). Linear models and empirical Bayes methods for assessing
differential expression in microarray experiments. Stat. Appl. Genet. Mol. Biol
3, Article3.
Stewart, S.A., Dykxhoorn, D.M., Palliser, D., Mizuno, H., Yu, E.Y., An, D.S.,
Sabatini, D.M., Chen, I.S., Hahn, W.C., Sharp, P.A., et al. (2003). Lentivirus-
delivered stable gene silencing by RNAi in primary cells. RNA 9, 493–501.
Storey, J.D., and Tibshirani, R. (2003). Statistical significance for genomewide
studies. Proc. Natl. Acad. Sci. USA 100, 9440–9445.
Sundararaman, B., Zhan, L., Blue, S.M., Stanton, R., Elkins, K., Olson, S., Wei,
X., Van Nostrand, E.L., Pratt, G.A., Huelga, S.C., et al. (2016). Resources for the
comprehensive discovery of functional RNA elements. Mol. Cell 61, 903–913.
Wheeler, J.R., Matheny, T., Jain, S., Abrisch, R., and Parker, R. (2016). Distinct
stages in stress granule assembly and disassembly. eLife 5, e18413.
Yang, D., Abdallah, A., Li, Z., Lu, Y., Almeida, S., and Gao, F.B. (2015). FTD/
ALS-associated poly(GR) protein impairs the Notch pathway and is recruited
by poly(GA) into cytoplasmic inclusions. Acta Neuropathol. 130, 525–535.
Yasuda, K., and Mili, S. (2016). Dysregulated axonal RNA translation in
amyotrophic lateral sclerosis. Wiley Interdiscip. Rev. RNA 7, 589–603.
STAR+METHODS

KEY RESOURCES TABLE

REAGENT or RESOURCE SOURCE IDENTIFIER

Antibodies
Rabbit polyclonal anti-G3BP1 MBL International Cat#RN048PW, RRID:AB_10794608
Mouse monoclonal anti-G3BP1 EMD Millipore Cat#05-1938, RRID:AB_10561767
Goat polyclonal anti-TIA1 Santa Cruz Biotech Cat#sc-1751, RRID:AB_2201433
Rabbit polyclonal anti-TARDBP Abnova Cat#H00023435-A01, RRID:AB_461752
Mouse monoclonal anti-VBP1 Origene Cat#UM500063, RRID:AB_2629076
Rabbit polyclonal anti-GABARAPL2 Genetex Cat#GTX102006, RRID:AB_1240874
Rabbit polyclonal anti-USP11 Bethyl Cat#A301-613A, RRID:AB_1211380
Rabbit polyclonal anti-PPIG Bethyl Cat#A302-075A, RRID:AB_1604291
Mouse monoclonal anti-Nestin EMD Millipore Cat#MAB5326, RRID:AB_2251134
Rabbit polyclonal anti-Isl1/2 Santa Cruz Biotech Cat#sc-30200, RRID:AB_2126589
Mouse monoclonal anti-SMI-31 EMD Millipore Cat#NE1022, RRID:AB_10690651
Chemicals, Peptides, and Recombinant Proteins
Dorsomorphin Tocris Cat#3093
SB431542 Tocris Cat#1614
Retinoic Acid Sigma Cat#R2625
SAG EMD Millipore Cat#566660
DAPT Tocris Cat#2634
CHIR 99021 Tocris Cat#4423
Purmorphamine Tocris Cat#4551
DMH-1 Tocris Cat#4126
Valproic acid Tocris Cat#2815
Puromycin Thermo Fisher Cat#A1113803
Thapsigargin Tocris Cat#1138
Deposited Data
Quantitative MS data This paper MassIVE repository (https://massive.ucsd.edu)
accession MSV000081554
SG core proteome dataset (Jain et al., 2016) N/A
Protein-protein interaction datasets for ATXN2, (Blokhuis et al., 2016) N/A
C9orf72 DPRs, FUS, OPTN, TDP-43 and UBQLN2
Protein-protein interaction datasets for TDP-43 (Freibaum et al., 2010) N/A
Protein-protein interaction datasets for (Lee et al., 2016) N/A
GR dipeptide repeats
Protein-protein interaction datasets for (Lin et al., 2016) N/A
PR dipeptide repeats
Human proteins with prion-like domains (March et al., 2016) N/A
Proteins co-precipitated with biotinylated isoxazole (Kato et al., 2012) N/A
Experimental Models: Cell Lines
Human Lenti-X 293T cells Clontech Cat#632180
Human HepG2 cells ENCODE Project N/A
Human HeLa-S3 cells Lécuyer Lab N/A
HEK293T G3BP1-APEX2-GFP This paper N/A
HEK293T hPGK-APEX2-GFP This paper N/A
CV-B iPSCs (Gore et al., 2011) N/A
CV-B G3BP1-APEX2-GFP This paper N/A
(Continued on next page)

Cell 172, 590–604.e1–e7, January 25, 2018 e1

Continued
REAGENT or RESOURCE SOURCE IDENTIFIER
hnRNPA2/B1 D290V-1.1 hiPSC Generated in-house N/A
(Martinez et al., 2016)
hnRNPA2/B1 D290V-1.2 hiPSC Generated in-house, N/A
(Martinez et al., 2016)
C9-3.2 G4C2 repeat-expansion hiPSC Generated in-house, N/A
fibroblasts described in
(Lagier-Tourenne et al., 2013)
C9-5.2 G4C2 repeat-expansion hiPSC Generated in-house, N/A
fibroblasts described in
(Lagier-Tourenne et al., 2013)
C9-6.3 G4C2 repeat-expansion hiPSC Generated in-house, N/A
fibroblasts described in
(Lagier-Tourenne et al., 2013)
Experimental Models: Organisms/Strains
D. melanogaster: strain w[1118] Gao Lab N/A
D. melanogaster: UAS-GFP strain Gao Lab N/A
D. melanogaster: UAS-(GR)80 transgenic (Yang et al., 2015) N/A
strain: Vg-Gal4/Cyo; UAS-(GR)80/TM6B
D. melanogaster: Mutant allele of Bel/DDX3X/Y Bloomington Drosophila BDSC:19945; Flybase: FBst0019945
y[1] w[67c23]; P{w[+mC] y[+mDint2] = EPgy2}bel[EY08943] Stock Center
D. melanogaster: RNAi of Bel/DDX3X/Y Bloomington Drosophila BDSC:28049; Flybase: FBst0028049
y[1] v[1]; P{y[+t7.7] v[+t1.8] = TRiP.JF02884}attP2 Stock Center
D. melanogaster: RNAi of Bel/DDX3X/Y Bloomington Drosophila BDSC: 35302; Flybase: FBst0035302
y[1] sc[*] v[1]; P{y[+t7.7] v[+t1.8] = TRiP.GL00205}attP2 Stock Center
D. melanogaster: Bloomington Drosophila BDSC: 18765; Flybase: FBst0018765
Mutant allele of Hrb27C/DAZAP1 Stock Center
w[1118];PBac{w[+mC] = WH}Hrb27C[f04375]/CyO
D. melanogaster: RNAi of Hrb27C/DAZAP1 Bloomington Drosophila BDSC: 31684; Flybase: FBst0031684
y[1] v[1]; P{y[+t7.7] v[+t1.8] = TRiP.JF01477}attP2 Stock Center
D. melanogaster: RNAi of Hrb27C/DAZAP1 Bloomington Drosophila BDSC: 31685;Flybase: FBst0031685
y[1] v[1]; P{y[+t7.7] v[+t1.8] = TRiP.JF01478}attP2 Stock Center
D. melanogaster: Mutant allele of Lig/UBAP2(L) Bloomington Drosophila BDSC: 18242 ; Flybase: FBst0018242
w[1118]; PBac{w[+mC] = RB}lig[e04268]/CyO Stock Center
D. melanogaster: Mutant allele of Lig/UBAP2(L) Bloomington Drosophila BDSC: 14943; Flybase: FBst0014943
y[1]; P{y[+mDint2] w[BR.E.BR] = SUPor-P}lig[KG08209]/ Stock Center
CyO; ry[506]
D. melanogaster: RNAi of Lig/UBAP2(L) Bloomington Drosophila BDSC: 61857; Flybase: FBst0061857
y[1] v[1]; P{y[+t7.7] v[+t1.8] = TRiP.HMJ23346}attP40 Stock Center
D. melanogaster: Mutant allele of Psi/FUBP1/3 Bloomington Drosophila BDSC: 27192 ; Flybase: FBst0027192
y[1] w[*]; P{w[+mC] = EP}Psi[G5862] Stock Center
D. melanogaster: RNAi of Psi/FUBP1/3 Bloomington Drosophila BDSC:31301; Flybase: FBst0031301
y[1] v[1]; P{y[+t7.7] v[+t1.8] = TRiP.JF01247}attP2 Stock Center
D. melanogaster: RNAi of Psi/FUBP1/3 Bloomington Drosophila BDSC: 31683; Flybase: FBst0031683
y[1] v[1]; P{y[+t7.7] v[+t1.8] = TRiP.JF01247}attP2 Stock Center
D. melanogaster: RNAi of Psi/FUBP1/3 Bloomington Drosophila BDSC: 34825; Flybase: FBst0034825
y[1] sc[*] v[1]; P{y[+t7.7] v[+t1.8] = TRiP.HMS00140}attP2 Stock Center
D. melanogaster: Mutant allele of Unr/CSDE1 Bloomington Drosophila BDSC: 17673 ; Flybase: FBst0017673
y[1] w[*]; P{w[+mC] = EP}Psi[G5862] Stock Center
D. melanogaster: Mutant allele of Unr/CSDE1 Bloomington Drosophila BDSC: 10757 ; Flybase: FBst0010757
w[1118]; PBac{w[+mC] = PB}Unr[c01923] Stock Center
(Continued on next page)

e2 Cell 172, 590–604.e1–e7, January 25, 2018

Continued
REAGENT or RESOURCE SOURCE IDENTIFIER
D. melanogaster: RNAi of Unr/CSDE1 Bloomington Drosophila BDSC: 32432; Flybase: FBst0032432
y[1] sc[*] v[1]; P{y[+t7.7] v[+t1.8] = TRiP.HMS00428}attP2 Stock Center
D. melanogaster: RNAi of Unr/CSDE1 Bloomington Drosophila BDSC: 29334; Flybase: FBst0029334
y[1] v[1]; P{y[+t7.7] v[+t1.8] = TRiP.JF02496}attP2 Stock Center
D. melanogaster: GMR-GAL4/CyO, tub-GAL80; (Periz et al., 2015) N/A
UAS-FUS-hR521C/TM6B, Tb
D. melanogaster: GMR-GAL4, UAS-TDP-43-hM337V/ (Periz et al., 2015) N/A
CyO, tub-GAL80
D. melanogaster: Rox8e04432 Exelixis Collectiion at PBac{RB}Rox8e04432; Flybase: FBst1015699
Harvard University
D. melanogaster: Rbp6d08411 Exelixis Collectiion at P{XP}Rbp6d08411; Flybase: FBst1011661
Harvard University
D. melanogaster: ligf03269 Exelixis Collectiion at PBac{WH}ligf03269; Flybase: FBst1018357
Harvard University
D. melanogaster: CG2889d07154 Exelixis Collectiion at P{XP}CG2889d07154 mapped
Harvard University internally; Flybase: FBst1011297
D. melanogaster: D12e01238 Exelixis Collectiion at PBac{RB} D12e01238: mapped
Harvard University internally; Harvard Only
D. melanogaster: Su(var)205c06825 Exelixis Collectiion at PBac{PB}Su(var)205c06825;
Harvard University Flybase: FBst1008382
D. melanogaster: shepd07053 Exelixis Collectiion at P{XP}shepd07053; Flybase: FBst1011268
Harvard University
Oligonucleotides
Oligos for PCR, cloning and siRNA, see Table S8 This paper N/A
Recombinant DNA
Plasmid: pcDNA3 APEX2-NES (Lam et al., 2015) Addgene #49386
Plasmid: GFP-Fusion HR Targeting Vector System Biosciences (SBI) Cat#HR120PA-1
Plasmid: HR_G3BP1-V5-APEX2-GFP This study N/A
Plasmid: hPGK_V5-APEX2-GFP This study N/A
Plasmid: pSpCas9(BB)-2A-GFP (PX458) Gift from Feng Zhang Addgene #48138
(Ran et al., 2013)
Plasmid: pLIX403_UBAP2L_mCherry This study N/A
Plasmid: pLIX403_DUBA_UBAP2L_mCherry This study N/A
Plasmid: pRSV-Rev Gift from Didier Trono Addgene #12253
(Dull et al., 1998)
Plasmid: pMDLg/pRRE Gift from Didier Trono Addgene #12251
(Dull et al., 1998)
Plasmid: pCMV-VSV-G Gift from Bob Weinberg Addgene #8454
(Stewart et al., 2003)
Software and Algorithms
Enrichr Gene Set Enrichment Analysis (Kuleshov et al., 2016) http://amp.pharm.mssm.edu/Enrichr/
Cytoscape (Shannon et al., 2003) http://www.cytoscape.org
Detecting significant changes in protein abundance (Kammers et al., 2015) http://www.biostat.jhsph.edu/kkammers/
software/eupa/R_guide.html

CONTACT FOR REAGENT AND RESOURCE SHARING

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact,
Gene W. Yeo (geneyeo@ucsd.edu). Important plasmids described in this study will be deposited in the Addgene plasmid respository
and available under a standard MTA.

Cell 172, 590–604.e1–e7, January 25, 2018 e3

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Immortalized human cell lines and human pluripotent stem cells (hiPSCs) were utilized in this study. The Lenti-X HEK293T cell line is
derived from human female tissue, the HepG2 cell line is derived from human male hepatocellular carcinoma tissue and HeLa S3 cells
are derived from human female cervical adenocarcinoma tissue. HEK293T and HeLa cells were maintained in DMEM and HepG2
cells in Hyclone growth medium both supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37 C in a humid-
ified incubator under 5% CO2. hiPSCs were maintained under feeder-free conditions in mTeSR1 medium (Stem Cell Technologies)
and propagated either by single-cell passaging using Accutase or clump-passaging using enzyme-free dissociation buffer (EDTA).
Flies were reared on standard yeast-agar-cornmeal medium and crosses were performed at 25 C. The degenerative eye phenotype
was assessed two weeks after the crosses were performed, while the wing margin notching phenotype was scored in 3-5 days old
adult flies of the F1 generation.

METHOD DETAILS

Generation and maintenance of neural progenitor cells

Small molecule neural progenitor cells (smNPCs) were grown in medium consisting of DMEM/F12+Glutamax, 1:200 N2 supplement,
1:100 B27 supplement, penicillin/streptomycin (Life technologies), 100mM ascorbic acid (Sigma, A4544), 3mM CHIR99021
(CHIR, Tocris 4423) and 0.5mM Purmorphamine (PMA) (Tocris 4551 and passaged using Accutase. Generation of smNPCs from
iPSCs was adapted from (Reinhardt et al., 2013). Briefly, human iPSCs at 70%–80% confluency were dissociated using accutase
and resuspended at 1x10^6 cells/ml in N2B27 medium (DMEM/F12+Glutamax, 1:200 N2 supplement, 1:100 B27 supplement,
150mM ascorbic acid and 1% Penicillin/Streptomycin) supplemented with 1mM Dorsomorphin, 10mM SB431542, 3mM
CHIR99021, 0.5mM Purmorphamine (PMA) and 5mM Rock inhibitor (Y-26732). 3 million cells were transferred into one well of an
uncoated 6-well tissue culture plate and incubated at 37 C, 5% CO2 on a shaker at 90rpm. Uniform small EBs formed within 24h
and increased in size over the following days. After 48h a full media change was performed with N2B27 medium supplemented
with D, SB, CHIR and PMA. At this time, about 2/3 of EBs were either discarded or EBs were split across 3 wells of a 6-well plate
to reduce the high cell density required initially to ensure uniform formation of embryoid bodies. On days 3-5, half medium changes
were performed with fresh N2B27 + D, SB, CHIR and PMA. On day 6, dorsomorphin and SB were withdrawn and a full medium
change with smNPC medium (N2B27 + 3mM CHIR + 0.5mM PMA) was performed. At this stage, neuroepithelial folds were clearly
visible in all EBs. On day 8, EBs were triturated by pipetting 10-15 times with a P1000 pipette and plated onto matrigel-coated
6-well or 10cm plates (1 well of a 6-well plate per 10cm plate). After 3-4 days, attached EB fragments and outgrown cells were
dissociated to single cells with accutase and split at a 1:6 to 1:8 ratio onto matrigel. After the first passage, cells were passaged
at a 1:10 to 1:15 ratio every 3-6 days. For the first few passages, large flat non-smNPCs could be observed between smNPC colonies,
but progressively disappeared no later than passages 3-6 in almost all cell lines.

Generation of iPSC-derived motor neurons

Motor neurons were differentiated from iPSCs as described in (Martinez et al., 2016). Briefly, human iPSCs were grown plated
into matrigel-coated 6-well plates or 10cm culture dishes to reach 70%–90% confluency in mTeSR1 medium within 2-3 days.
On day 1 of the differentiation protocol, medium was changed to N2B27 medium (DMEM/F12+Glutamax, 1:200 N2 supplement,
1:100 B27 supplement, 150mM ascorbic acid and 1% Penicillin/Streptomycin) supplemented with 1mM Dorsomorphin, 10mM
SB431542, 3mM CHIR99021. Cells were maintained with daily medium changes in the same medium for 6 days. On day 7,
medium was changed to N2B27 medium (DMEM/F12+Glutamax, 1:200 N2 supplement, 1:100 B27 supplement, 150mM ascor-
bic acid and 1% Penicillin/Streptomycin) supplemented with 1mM Dorsomorphin, 10mM SB431542, 200nM Smoothened Agonist
(SAG) and 1.5uM retinoic acid (RA). Medium was changed daily with increasing volumes to adjust for cell density until day 18. At
day 18 of differentiation, cells were dissociated using Accutase and either plated directly for continued differentiation or
optionally expanded in motor neuron progenitor (MNP) medium as described in (Du et al., 2015). Optionally, after dissocation,
cells were plated onto matrigel-coated 10cm plates at a density of 3-5 million cells per plate into N2B27 medium supplemented
with 3 mM CHIR99021, 2 mM DMH1, 2 mM SB431542, 0.1 mM RA, 0.5 mM Purmorphamine and 0.5mM valproic acid (VPA). Cells
were maintained for no more than 5 passages under these conditions with weekly splitting using Accutase at 1:8-1:12 before
final differentiation. For continued differentiation, cells were plated at a density of 10 million cells per plate into 10cm
plates plate serially coated with 0.001% ( = 0.01mg/ml) poly-D-lysine (PDL, Sigma, P6407) and poly-L-ornithine (PLO, Sigma,
P3655) followed by 20ug/ml laminin (Life technologies, 23017015). Cells were plated into N2B27 medium supplemented
with 200nM SAG and 1.5uM RA and 10uM rock inhibitor. Medium was changed on day 20 and cells transferred into
N2B27 medium supplemented with 2uM DAPT on day 22. For imaging, cells were dissociated again at day 23 and plated
into PDL/PLO/laminin-coated 96-well plates. Medium as changed into N2B27 medium without additional small molecules on
day 25 and cells were maintained with medium changes every 2-3 days thereafter. Cells were stressed and fixed between
day 29-32 of differentiation.

e4 Cell 172, 590–604.e1–e7, January 25, 2018

Plasmid construction
To generate the donor vectors used to tag the endogenous G3BP1 locus in human cells, we modified the HR120PA-1 targeting vector
(System Biosciences (SBI)) by replacing GFP with an APEX2-(GGGGS)2-GFP fusion protein. 1.5kb arms of homology were ampli-
fied from genomic DNA using primers that introduced 35bp overhangs with the targeting vector template on each end. The final
G3BP1-targeting vector was assembled by Gibson assembly. G3BP1-APEX2-GFP was co-transfected with Cas9 expression vector
px458 (gift from Feng Zhang, Addgene plasmid #48138) into HEK293T cells using lipofectamine 2000 or electroporated into CV-B iPS
cells using an Amaxa Nucleofector with Stem Cell Kit 1 and pulse setting B-016. 48-72h post-transfection, puromycin was added to
the medium at 1ug/ml and cells kept in selective medium for 2-4 days. After 10-14 days, homogeneously green fluorescent single-cell
derived colonies were manually picked under a stereomicroscope, expanded and tested for APEX2 activity.
For UBAP2L overexpression experiments, full-length and DUBA-UBAP2L-mCherry fusion constructs were cloned into pLIX_403
(gift from David Root, Addgene plasmid # 41395) and packaged into lentiviral particles. MNPs were transduced and selected with
2mg/ml puromycin (Life technologies, A1113803) for 7 days starting 2 days post-transduction. Expression was induced by adding
100ng/ml doxyxycline for 24h. To induce SG formation, cells were treated with 250mM (NPCs, MNs) or 500mM (HEK293T, HeLa,
HepG2 cells) NaAsO2 for 30min (HeLa cells) or 1h (NPCs, MNs, HEK293T and HepG2 cells). Alternatively, SG formation was
induced by treatment with 10ug/ml puromycin for 24h (MNs), 500nM thapsigargin (NPCs) or by heat shock for 30min at 45 C
(HeLa, HepG2 cells).

SILAC labeling with isotopically modified amino acids

For SILAC experiments, DMEM without L-arginine and L-lysine (Pierce catalog no. PI88420) was supplemented with 10%
dialyzed FBS (Pierce, PI88440), penicillin/streptomycin, and 0.4mM and 0.8mM, respectively, of either unlabeled L-Lysine:HCL
and L-Arginine:HCl (Sigma, cat no. L8662 and A6969) or isotopically labeled L-Lysine: 2HCl (13C6, 15N2) and L-Arginine:HCl (13C6,
15
N4) (Cambridge Isotope laboratories, cat no. CNLM-291 and CNLM-539). Both heavy and light medium were additionally supple-
mented with 200mg/ml L-Proline (Sigma, cat no. P5607).
For SILAC labeling of smNPCs, DMEM/F12 without L-arginine and L-lysine (Pierce catalog no. PI88215) was used instead of
regular DMEM/F12 and supplemented with 0.7mM and 0.5mM, respectively, of either unlabeled L-Lysine:HCL and L-Arginine:HCl
(Sigma, cat no. L8662 and A6969) or isotopically labeled L-Lysine: 2HCl (13C6, 15N2) and L-Arginine:HCl (13C6, 15N4) (Cambridge
Isotope laboratories, cat no. CNLM-291 and CNLM-539).

APEX-mediated biotinylation
HEK293Ts and NPCs were grown in heavy or light SILAC medium for at least 5 passages prior to APEX labeling and isotope label
incorporation efficiency was confirmed to be above 98%. Cells were seeded in 10cm culture dishes one day prior to labeling to
be 80% confluent the following day and either left unstressed or treated with either 250mM (NPCs) or 500mM (HEK293T) NaAsO2
or 500nM thapsigargin for 1h at 37 C. 500mM biotin-phenol (BP) was added to the medium at the same time as stressors except for
the no-substrate control samples. APEX labeling was performed by adding hydrogen peroxide to a final concentration of 1mM for
60 s before quenching the biotinylation reaction by adding Trolox ((+/ )-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic
acid, Sigma 238813) and sodium L-ascorbate (Sigma A4034) to a final concentration of 5 and 10mM, respectively. Samples were
washed once with cold PBS, collected using cell scrapers, pelleted for 3min at 300 g and immediately suspended in cold lysis buffer
(8M urea, 150mM NaCl, 20mM Tris-HCl pH 8.0, Protease Inhibitor Cocktail Set III, EDTA-Free (EMD Millipore, cat no. 539134), 5mM
Trolox and 10mM sodium L-ascorbate). Samples were sonicated and cleared by centrifugation at 12000rpm for 10min at 4 C. Protein
concentration was determined using by 660nm protein assay (Pierce, PI22660) and equal amounts of protein from corresponding
light and heavy labeled samples were mixed for a total of 2-4mg of protein. Samples were diluted to 2M urea by adding 3 volumes
of 150mM NaCl, 20mM TrisHCl pH 8.0 with protease inhibitors and quenchers. For affinity purification, 100ul of streptavidin mag-
netic beads (Pierce, PI88817) were washed once in 2M urea buffer, resuspended directly in the sample, incubated for 2h at room
temperature and washed 8 times in 2M urea buffer. Following the washes, beads were centrifuged at 240 RCF for 5 min at 4 C.
The supernatant was removed and a volume of 50mM Ammonium bicarbonate buffer equal to the volume of the beads was added.
For the on-bead digestion of the IP samples, the ammonium bicarbonate buffer was removed and replaced with an equal volume of
20mM Tris pH8.0 with endoproteinase Lys-C (Wako) at a 1:100 (w/w) enzyme substrate ratio. Samples were incubated for 1hr at
37 C. Following the Lys-C digestion, CaCl2 was added to a final concentration of 1mM along with 500ng sequencing grade trypsin
(Promega). The corresponding input samples for each IP were diluted to a final urea concentration of 1M using 50mM Ammonium
bicarbonate. Lys-C digestion was done as described above for the IP samples followed by trypsin digestion with a 1:100 (enzyme:
protein) ratio After trypsin addition, all samples were incubated at 37 C for overnight with agitation. After the digestion, an equal
volume of 5% formic acid was added to the digestion mixture and incubated at room temperature for 10 minutes. The supernatant
was transferred to a new 1.5mL tube and the elution step was repeated one more time. The trypsin-digested input and IP samples
were concentrated and desalted using the Stage-Tip method and reconstituted in a 5% Formic acid/5% acetonitrile for MS analysis.

IF, imaging and image analysis

Cells were fixed for 20 min in 4% formaldehyde, 1X PBS, followed by permeabilization for 10 min with 0.5% Triton, 1X PBS. Cells
were rinsed with 1X PBS and blocked with blocking buffer (1X PBS, 2% BSA, 0.02% Triton). Cells were incubated with the primary

Cell 172, 590–604.e1–e7, January 25, 2018 e5

antibodies against SG marker like TIA1 (TIA1, dilution 1:100, cat.# sc-1751,Santacruz) and antibodies against RBPs Sundararaman
et al., 2016) diluted in blocking buffer for 2 hour at room temperature or overnight at 4 C. Then, the cells were thoroughly washed with
1X PBS, 0.2% Tween 20, and incubated for 2 hour with secondary antibodies (Alexa Fluor 647, cat. # A21447, Alexa Fluor 594, cat. #
A21207, Life technologies and Alexa Fluor 488, cat. # 111-546-144, JacksonImmuno, dilution 1:500) diluted in blocking buffer. Cells
were washed, incubated for 5 min with DAPI and washed again. Cells were stored in the dark at 4 C in 1X PBS or 50% glycerol/PBS
for long-term storage. All images were taken using high content screen microscopy, ImageXpress Micro.

Drosophila genetics
Flies were reared on standard yeast-agar-cornmeal medium and crosses were performed at 25 C. Drosophila transgenic
strains carrying GAL4 inducible human ALS disease causing alleles of FUS/TLS and TDP-43 were previously described
(Lanson et al., 2011; Ritson et al., 2010). Standard genetic procedures were used to generated the GMR-GAL4/CyO, tub-GAL80;
UAS-FUS-hR521C/TM6B, Tb and GMR-GAL4, UAS-TDP-43-hM337V/CyO, tub-GAL80 transgenic strains (Periz et al., 2015).
Drosophila strains containing the Exelixis insertional disruptions are publically available from the Department of Cell Biology, Harvard
Medical School include Rox8e04432, Rbp6d08411, ligf03269, CG2889d07154, D12e01238, Su(var)205c06825 and shepd07053. The dominant
effect of the introduction of these inserts on degenerative eye phenotypes of GMR-GAL4; UAS-FUS-hR521C and GMR-GAL4,
UAS-TDP-43-hM337V was assessed two weeks after the crosses were performed. Qualitative changes in pigmentation, ommatidial
structure and glossiness phenotypes were monitored for enhancement or suppression.
UAS-(GR)80 transgenic fly lines were generated previously (Yang et al., 2015). Vg-Gal4/Cyo; UAS-(GR)80/TM6B flies were crossed
with individual genetic mutant or UAS-RNAi lines for a specific gene, which were obtained from the Bloomington Drosophila Stock
Center. For crosses with genetic mutant alleles, w1118 flies were used as the control. For crosses with UAS-RNAi lines, UAS-GFP
served as the control. After the cross, 3-5 days old adult flies of the F1 generation were scored under the dissecting microscope.
The number of flies with or without the wing margin notching phenotype was counted.

Protein interaction network analysis

To retrieve protein interaction data and build protein-protein interaction networks, we queried the Proteomics Standard Initiative
Common QUery InterfaCe (PSICQUIC) web portal (http://www.ebi.ac.uk/Tools/webservices/psicquic/view/main.xhtml) for PPI
data form the mentha, IntAct and MINT databases. We restricted results to only human interactors that had been experimentally
validated in AP-MS experiments (i.e., search terms MI:0006: anti bait coimmunoprecipitation and MI:0007: anti tag coimmunopre-
cipitation). The resulting data were combined with the most recently available dataset based on AP-MS interactions of 5000 bait
proteins from the Bioplex website (http://bioplex.hms.harvard.edu). We used Cytoscape to visualize the resulting PPI dataset
consisting of 14,352 nodes and 102,551 non-redundant edges. We extracted PPI data for 361 SG proteins and used the Prefuse
Force Directed Layout to visualize the network. The internal Cytoscape Network Analyzer plugin was used to calculate and visualize
network parameters.

Protein domain and gene ontology analysis

Domain analysis was done by retrieving PFAM domains through the NCBI Conserved Domains Database (https://www.ncbi.nlm.nih.
gov/Structure/cdd/cdd.shtml). Low complexity domains and intrinsically disordered regions were calculated as previously described
(Beckmann et al., 2015; Conrad et al., 2016). Gene ontology enrichment analysis and PPI hub analysis was performed through the
Enrichr Gene Ontology enrichment tool (http://amp.pharm.mssm.edu/Enrichr/) (Kuleshov et al., 2016). Results were ranked by the
‘combined score’, which combines p value and z-score by multiplication: c = log(p) * z.

QUANTIFICATION AND STATISTICAL ANALYSES

MS data collection and analysis

Samples were analyzed in triplicate using a Q-Exactive mass spectrometer (Thermo Scientific, San Jose, CA) with essentially
the same nHPLC and instrument method as described previously (Gendron et al., 2016) with the following modifications: For input
samples, peptides were eluted using a 60 min ACN gradient (45 minute 2%–30% ACN gradient, followed by a 5 minute 30%–60%
ACN gradient, a 2 minute 60%- 95% gradient, with a final 8 minute isocratic step at 0% ACN) at a flow rate of 250 nl/min. A dynamic
exclusion time of 20 s was used and singly charged ions, charge states above 6 and unassigned charge states were excluded. For IP
samples, peptides were eluted using a 120 min ACN gradient (100 minute 2%–30% ACN gradient, followed by a 5 minute 30%–60%
ACN gradient, a 5 minute 60%- 95% gradient, with a final 10 minute isocratic step at 0% ACN) at a flow rate of 250 nl/min. A dynamic
exclusion time of 40 s was used and singly charged ions, charge states above 6 and unassigned charge states were excluded. The
resultant RAW files were analyzed using Andromeda/MaxQuant (version 1.6.0.16) (Cox and Mann, 2008). Data were searched against
a concatenated target-decoy database comprised of forward and reversed sequences from the reviewed UniprotKB/Swiss-Prot
FASTA Human database (2015) with GFP and common contaminants appended (22,000 entries). Arg10 and Lys8 were selected
as isotope labels and trypsin was specified for protein digestion. Variable modifications for methionine oxidation and protein
N terminus acetylation and a fixed modification for cysteine carbamidomethylation were allowed. A mass accuracy of ± 50ppm
was specified for the first search and ± 4.5ppm for the main search. A maximum of 2 missed cleavages and 5 modifications were

e6 Cell 172, 590–604.e1–e7, January 25, 2018

allowed per peptide and the maximum charge was set to 7. The minimum allowed peptide length was 7 amino acids and matching
between runs was enabled for data obtained from the same cell line. The data were filtered using protein, peptide and site level false
discovery rates of 0.01. Unique and razor peptides were used for quantification. Matches to common contaminants, reverse iden-
tifications and identifications based only on site-specific modifications were removed prior to further analysis.
To determine log2 H/L ratio cutoffs, proteins in each experimental replicate were ranked by descending log2H/L ratios and the
fraction of known SG proteins in a rolling window (size = 200) was calculated. A cutoff was determined to be the point at which
the frequency of known SG proteins fell below 2 times the background frequency. For each experimental design in HEK293T cells,
proteins with log2 H/L ratios above the cutoff in at least 2/3 (Exp. 2-4) or all 3 replicates (Exp.1) were retained as candidates. A final list
of 123 candidate SG proteins in HEK293T cells was assembled from all hit candidates in Exp. 3 (stressed G3BP1-APEX2-GFP versus
stressed NES-APEX2-GFP) that overlapped with hit candidates from Exp.1 (stressed G3BP1-APEX2-GFP versus unstressed
G3BP1-APEX2-GFP) or Exp. 2 (stressed G3BP1-APEX2-GFP versus stressed G3BP1-APEX2-GFP without biotin phenol).
In parallel, individual peptide intensities were analyzed by an empirical Bayes approach (Kammers et al., 2015)using an adaption
of the original R script (http://www.biostat.jhsph.edu/kkammers/software/eupa/R_guide.html) suitable for SILAC data. Briefly,
individual heavy and light peptide intensities were log2-transformed and protein-level intensities were calculated from the
median of all peptides identified and quantified for each protein. Global median intensity levels were normalized and a moderated
two-sample t test was performed, comparing heavy and light intensities for each protein. The resulting moderated p values are
corrected for multiple hypothesis testing using a modified Benjamini-Hochberg false discovery rate (FDR) approach to determine
a moderated q-value (q.mod) (Storey and Tibshirani, 2003). Proteins identified at a false-discovery rate (moderated q-value) of
0.05 were annotated as significantly different in abundance.
For neural progenitor cells (NPCs), we only performed Exp.1 and 2 in biological duplicates, but using two different stressors
(NaAsO2 or thapsigargin) for each experiment. To identify candidate SG proteins in NPCs, we determined log2 H/L ratio cutoffs
for each experimental replicate using an identical procedure to that used in HEK293T cells. For each stressor, a candidate list
was compiled from proteins with ratios above the cutoff in 3 out of 4 combined replicates of Exps. 1 and 2. The resulting lists
were combined to yield an exploratory list of 178 NPC SG candidates. To obtain sufficient power for statistical testing, we combined
all 4 replicates of each experimental design obtained with the two stressors. Only proteins with peptide intensity measurements
across all 4 replicates were considered in the analysis, somewhat limiting the number of proteins for which statistical significance
could be assessed.

Image analysis
MetaXpress v3.1 software was used for all image analysis and quantifications were carried out using an in-house script
(see Methods S1).

DATA AND SOFTWARE AVAILABILITY

The accession number for the proteomics data reported in this paper is MassIVE MS data repository (https://massive.ucsd.edu/
ProteoSAFe/static/massive.jsp): MSV000081554.

Cell 172, 590–604.e1–e7, January 25, 2018 e7

Supplemental Figures

Figure S1. APEX Cell Line Generation, MS Experimental Design, and Data and Analysis, Related to Figure 1
(A) Schematic of CRISPR-Cas9-mediated endogenous tagging of the G3BP1 locus.
(B) Schematic of generating a constitutive hPGK::NES-APEX2-GFP-expressing HEK293T cell line.
(C) Schematic description of the SILAC experimental workflow.
(D) Venn diagram showing overview of all proteins detected by MS in streptavidin affinity-purified samples, corresponding input samples in relation to a list of
known SG proteins.
(E) Flow chart depicting data analysis steps to identify candidate SG proteins.
Figure S2. Reproducibility of Protein Identification and Quantitation across Replicates, Related to Figure 1
(A) Venn diagrams showing overlap between proteins identified from HEK293T cells in biological replicate experiments.
(B) Scatterplots showing correlation between log2 H/L ratios for identified proteins from biological replicate experiments.
Figure S3. APEX-Mediated Biotinylation, Experimental Design, and Detected Proteins in Neural Progenitor Cells, Related to Figure 3
(A) Streptavidin staining of unstressed (top panel) and sodium arsenite-treated (middle and bottom panels) CV-B G3BP1-APEX2-GFP neural progenitor cells.
Cells were either incubated in the presence (upper and middle panels) or absence (lower panel) of biotin phenol.
(B) SG-APEX experimental designs used in NPCs.
(C) Venn diagram showing overview of all proteins detected by MS in NPC input and IP samples and overlap with known SG proteins.
(D) Venn diagram showing overlap of all proteins detected above fold-change cutoff in NPC stressed with either sodium arsenite or thapsigargin.
Figure S4. Expression of Cell-Type-Specific Markers and Neurite-Localized Granules in iPSC-Derived Motor Neurons, Related to Figure 5
(A) IF staining of wild-type and HNRNPA2B1 mutant motor neurons showing expression of the motor neuron-specific phosphorylated neurofilament SMI-31 and
the transcription factor ISL1/2.
(B) IF staining of wild-type and HNRNPA2B1 mutant motor neurons that were either left untreated or stressed with puromycin, then co-labeled with G3BP1 (green)
and a panel of RBP antibodies (red). Upper panels are merged views with lower resolution. In each panel, the indicated insets at the bottom are zoomed views of
the same field showing G3BP1 (green) and the RBP (red).
(C) Enrichment analysis for KEGG pathways and Biological Process Gene Ontology term as determined by the Enrichr gene enrichment analysis tool.
Figure S5. Extended Dataset Cross-Comparison, Additional Fly Modifiers, UBAP2L Protein Structure and Co-localization with Stress
Granule Proteins, Related to Figure 6
(A) Venn diagram showing overlap between SG proteins identified in our study, compared to the SG core proteome (Jain et al., 2016) and known SG proteins.
(B) Heatmap for all 1312 proteins represented across selected SG and neurodegeneration-relevant datasets, indicating whether a protein is present (blue box) or
absent (white box) from each dataset. Proteins are ranked by the number of datasets they are part of in descending order from top to bottom.
(C) Quantitation of the wing notching phenotype caused by poly(GR) toxicity in flies. w1118 flies were used as the control for genetic mutant alleles, while UAS-GFP
served as the control for different UAS-RNAi lines. The Bloomington stock numbers for each mutant or RNAi line are listed.

(legend continued on next page)

(D) IF images of 293FITR cells with inducible expression of either a full-length UBAP2L-mCherry fusion protein (top) or a truncated UBAP2L-mCherry fusion
protein missing the N-terminal UBA domain (bottom).
(E) Graphs showing protein domains, distribution of low complexity domains and intrinsically disordered regions across the length of human UBAP2L protein.